High Performance Liquid Chromatography PDF

High Performance Liquid Chromatography Principle, Theory, Instrumentation and Application Dr. A. Amsavel, M.Sc., B.Ed., Ph.D.  An Overview Introduction Chromatographic Separation Definitions HPLC Techniques Instrumentation & Types of detectors Qu...

High Performance Liquid Chromatography Principle, Theory, Instrumentation and Application Dr. A. Amsavel, M.Sc., B.Ed., Ph.D.  An Overview Introduction Chromatographic Separation Definitions HPLC Techniques Instrumentation & Types of detectors Qualification & Calibration Method Development – Basics Column Chemistry Applications, Specifically Pharmaceutical QC  What is Chromatography? Chromatography is a separation technique used for Qualitative and Quantitative Analysis. Essential for testing of chemicals in an Industries & academics Technique developed in 1941 by Martin & Synge and were awarded Nobel Prize in 1952 (partition chromatography) In 1951 first GC experiment was performed by Martin & James Horvath & Lipsky built the first high-pressure liquid chromatograph. End of the 70’s improvised – High Performance Liquid Chromatograph instruments become familiar. Pharmaceutical industry made HPLC the workhorse beginning in the 1970s and become boom in 1980s Since 2006 advance models like UPLC, RRLC,UFLC, RSLC.. are available  Chromatographic Separation Chromatography is based on a Physical Equilibrium that results when a solute is transferred between the mobile and a stationary phase. K = Distribution coefficient or Partition ratio K = CS/ CM CS -Molar conc. of the solute in the stationary phase CM -Molar conc. of the solute in the mobile phase.  Separation in Column In a mixture, each component has a Different distribution coefficient in liquid Stationary phase and Mobile phase. The sample then has the opportunity to interact with the stationary phase as it moves past it. Samples/ molecules that interact greatly, which move slowly and weekly interact are move quickly. Because of this difference in rates, the sample is Separated into their components. “Like Attracts Like – Opposites are Not Attracted”  Definitions Gradient elution: Continuously changing the solvent composition during the chromatographic run is called gradient elution or solvent programming. Retention factor (k):1 Also known as the “capacity factor (k)” k = time spent by substance in Stationary phase time spent by substance in mobile phase k = (tR - tM) /tM Hold-up time (tM): The time required for elution of an unretained component Retention time (tR) ,  Definitions  Number of theoretical plates (N): A measure of column efficiency. For Gaussian peaks, it is calculated by: N = 16(tR/W) 2 where tR is the retention time of the substance, &W is the peak width at base  Resolution (RS): The resolution is the separation of two components in a mixture, calculated between peaks (1&2); tR - Retention time & W – peak width RS = 2 × (tR2 - tR1)/(W1 + W2)  Symmetry factor (AS): Also known as the “tailing factor”, of a peak is calculated by: AS = W0.05/2f (Fig-1) Where W-0.05 is the width of the peak at 5% height from base and f is the distance from the peak maximum to the leading edge of the peak. Fig-1 Signal-to-noise (S/N) ratio: Signal is useful system suitability parameter. (Sensitivity) The S/N ratio is S/N ratio = 2H/h ) Noise  HPLC Techniques Polarity 1. Normal Phase 2. Reversed Phase Charge 3. Anion Exchange (SAX, WAX) 4. Cation Exchange (SCX, WCX) Size 5. Size Exclusion Chromatography (SEC) 6. Gel Permeation Chromatography (GPC)  Normal Phase HPLC Polar stationary phase and non-polar mobile phase. Stationary phase is usually Silica, Cyanopropyl- bonded, Amino-bonded etc.. Chiral columns Mobile phases are Hexane, Heptane, Methylene chloride, chloroform, diethyl ether , ethyl acetate, THF or mixture Polar samples are retained on the polar column longer than less polar & non-polar samples. Used for: Water-sensitive compounds / geometric isomers / cis-trans isomers/chiral compounds.  Reverse Phase HPLC Reverse Phase is opposite to normal phase: Stationary phase is nonpolar ( C-18 Hydrophobic) and Mobile phase is a polar liquid; eg. mixtures of water and methanol or acetonitrile. Lowering the amount of organic solvent in the mobile phase increases the retention time. Retention is based on Van dar waal’s Interaction between the non- polar stationary phase and the molecule (Fig) Nonpolar samples are retained longer than Neproxen polar molecules. Over 90% of chromatographers use RPC The technique is used for non-polar, polar, ionizable and ionic molecules …  Separation and Polarity Chromatographic Retention Behavior “Like Attracts Like – Opposites are Not Attracted” Polars attracted to other Polars Non-polars attracted to other non-polars Polarity Compound Mobile Phase- Coulmn Solvent Polar Salts Water Diol Acids Alcohols Amine Alcohols Acetonitrile Silica Ketones Ethers Ethylacetate Cyano Halogenated THF Phenyl compounds Dichloromethane C8 (Octyl) Non- Aliphatic Toluene C18 (Octadecyl) Polar hydrocabons Hexane  Ion-pair chromatography Ion-pair chromatography is a subset of reversed-phase chromatography. Used for separation of complex mixtures of very polar and ionic molecules. Organic salt containing a large organic counter-ion, such as a quaternary ammonium ion or alkyl sulfonate is added to the mobile phase as an ion-pairing reagent. Eg. Heptane or Octyl sulfonic acid (for base) Tetrabutyl ammonium Hydroxide, certrimide (for acid) Ion-pairing reagents having a charge opposite to the analyte of interest as well as a substantial hydrophobic region that allows interaction with the stationary phase, plus associated counter-ions. This counter ion forms an uncharged ion pair with a solute ion of opposite charge in the mobile phase. This ion pair then partitions into the nonpolar stationary phase giving differential retention of solutes based on the affinity of the ion pair for the two phases.  Ion-pair chromatography Advantage of Ion-pair chromatography : Reduced separation times; Highly reproducible results; Sharper peak shapes; Simultaneous separation of ionized and non-ionized analytes; and Wide choice of additives to improve separation Example for Ion-pairing reagent (IPR) and interaction with molecule Cationic ion-pairing agent : Ascorbic acid Anionic ion-pairing agent: Adrenaline with anion-pairing agent in ODS column. with an ion-pairing agent in ODS column. 0.1 M sodium acetate buffer pH 4.2, 0.1 M sodium phosphate buffer/methanol Acetonitrile 95:5 containing 0.03 M 9:1 containing 0.02 % sodium cetrimide (IPR) octanesulphonic acid (IPR)  Ion-Exchange HPLC The stationary phase is Ionically charged functional groups on polymer The mobile phase is an aqueous buffer, where both pH and ionic strength are used to control elution time. The mobile phase is an aqueous buffer (e.g. phosphate, formate, etc.). Opposite charge of the sample ions are attracted and elutes later. This technique is used almost exclusively with ionic or ionizable samples.  Ion-Exchange Mechanism In ion exchange, the column packing contains ionic groups (Eg. Sulfonic acid , tetraalkyl ammonium ) Useful for separation of inorganic and organic anions and cations in aqueous solution. Used to test Ionic dyes, amino acids, and proteins etc Type of Exchanger Exchanger Group Functional Cation Sulfonic acid- SO3-, Strong acid Carboxylic acid Weak acid -COOH, Anion Quaternary ammonium Strong base ion -NR3+ Weak base Amine group -NH2  Size Exclusion HPLC No interaction between the sample compounds and the column. The column is filled with material having precisely controlled pore sizes. Molecule /particles are separated according to its their molecular size. Molecules larger than the pore opening do not diffuse into the particles, while molecules smaller than the pore opening enter the particle and are separated. Large molecules elute first. Smaller molecules elute later. Mainly for polymer characterization and for proteins.  SEC Retention Mechanism Molecular Size in solution Analytes are dissolved in solution, injected into mobile phase Analytes are separated by their size once they are in solution There are two modes: Non-aqueous SEC [Gel Permeation Chromatography (GPC)] – Used in polymer separations Aqueous SEC [Gel Filtration Chromatography (GFC)]. – Used in biomolecule separations.  HPLC Instrumentation HPLC consists: 1. Reservoir containing the mobile phase, 2. Pump to force the mobile phase through the system at high pressure, 3. Injector to introduce the sample into the mobile phase 4. Chromatographic column, 5. Detector 6. Data collection device. HPLC–Typical Depiction  HPLC : Pump The role of the pump is to force the mobile phase at a specific flow rate, (mL/min). Normal flow rates in HPLC are in the 1- to 2-mL/min range. Typical pumps can reach pressures in the range of 400 to 600 bar. During the chromatographic experiment, a pump can deliver a constant mobile phase composition (isocratic) or an increasing mobile phase composition (gradient). Quaternary pump (low pressure) and binary pump (high pressure) are available. A flow rate range of 1–2 mL/min on a 4.6 mm dia. column will translate to a flow rate of ∼ 0.48 - 0.96 μL/ min for a 0.1 mm dia.  HPLC Pump Requirement The low flow rates obey Poiseuille’s law. A flow rate range of 1–2 mL/min on a 4.6 mm dia. Columns packed with 3- and 5-μm silica-based particles required < 400 bar (5800 psi) pressure in HPLC. Pressure will increase to maintain same flow rate at lower particle size. Pressure needed is inverse to the square of the particle diameter. Eg Pressure required at a given flow rate is 1100% higher when the column is packed with 1.5 μm particles than with 5 μm particles. UHPLC operates with microbore column and pump pressure designed to1300 bar (19,000 psi) and UPLC built for > 600bar Flow rate of ∼ 0.48 - 0.96 μL/ min for a 0.1 mm dia.  Mobile Phase Elution Isocratic (ISO means Same) Solvent Composition remains Same during the Entire Run.Eg. 60:40 Alcohol: Water Gradient Solvent Composition Changes throughout the HPLC Run time. Gradually Changed or Step Changes o Analysis time is reduced by proper composition & time o Achieve good Peak Separation/ Resolution & Peak shapes and Faster analysis of complex molecules.  Mobile phase Mobile phase should be thoroughly degassed to remove all dissolved gasses. Dissolved gas can be removed from solution by: Bubbling with helium Sonication / Vacuum filtration If the mobile phase is not degassed, air bubbles can form in the high-pressure system resulting in problems with system instability, spurious baseline peaks etc. Do not use Online degasser while THF as mobile phase due to degradation of Teflon in vacuum chamber.  HPLC: Injector The injector function is to introduce the sample into Column along with mobile phase. Multiport value The injector must also be able to withstand the high pressures of the liquid system. Mostly sample volumes used is 5 to 100 µL. Autosampler is available for automatic injection to analyze many samples continuously by setting the program in the system Fill the vials with sample and keep in the order of injection in auto sampler tray (100 samples) to inject automatically – measures the appropriate sample volume, – injects the sample automatically, – then flushes the injector to be ready for the next sample and continue all sample vials until all are processed …  HPLC Columns Considered the “heart of the chromatograph” High-purity spherical silica particles with low in trace metal content, of 2-10 μm diameter particles are coated with chemical stationary phase. Pore sizes of the particles are 60–150, 200–300, and 1,000–4,000°A, used for separation of small molecules, polypeptides/proteins, and very high molecular weight proteins respectively to allow the analyte to penetrate the pores. Nonporous packings- of very small silica particle 4.6 mm The below types of columns are advantage of speed and minimal solvent consumption and high plates > 100,000 plates/m – Microcolumn: Length 100 – 150 mm & ID 1.0 - 4.6-mm, PS.3 to 5µm – Capillary: various lengths, ID 0.1 - 1.0 mm; – Nano (i.d. < 0.1 mm, or sometimes stated as < 100 µm)  Guard Column & Ghost-Buster Column Guard column, is a short column packed with a similar stationary phase as the analytical column. The purpose of the guard column is to prevent impurities, such as highly retained compounds and particulate matter, system debris from reaching and contaminating the analytical column. In gradient elution unexpected peaks ie Ghost Peaks may appear. Ghost-Buster Column absorb the week polar and non-polar impurities in the mobile phase and eliminate ghost. Also baseline drift /fluctuation caused in the gradient is eliminated and gives stable baseline. It is connected between mixture and sample injection.  Column Oven Temperature Control in HPLC: Uniform temperature throughout the run give reproducible result. Column oven available with 5°C to 85°C. Reproducibility: Retention of molecule is also temperature dependent If temperature varies, the peak areas/heights may vary for the specific compounds. Peaks elutes faster if temperature is high (Ref Fig). Solubility If compounds are low solubility in the mobile phase, in the flow stream they may precipitate or forms salt in the column, by Increase the temperature or maintain the column temperature high to overcome Stability: 25°C Some of biological compounds such as enzymes or proteins, may not be stable at room 30°C temperature or higher. The temperature needs 35°C to be much lower down to 4°C  Detectors Detectors sense the separated components and provide a signal. Selection of detectors based on the compounds All the detectors are either concentration-dependent or mass dependant. Can connect multiple detectors for good response Commonly used detectors are;  UV-spectroscopy: Dual wavelength & PDA  Refractive Index  Fluorescence,  Mass Spectrometric  Electrochemical detectors  ELSD and etc  UV Detector  A modified UV spectrophotometer equipped with a flow cell.  UV light at selected wave length(s) is passes through a flow cell  If a compound elutes from the column that absorbs this light energy,  Absorbed energy is detected by the sensor, which converts as electrical signal, which is amplified and directed to data system.  Chromatogram is a plot of Variable Wavelength Detector absorbance as a function of elution time (RT)  UV absoption is based on the chromaphores in the molecule  Energy absorbed is proportional to the amount of component  The flow cell has a volume of 1–10 µL and a path length of 0.2–1 cm  Diode Array Detector  This is working in the same principle of UV detector,  In UV only single or dual wave length is absorbed, but in a diode array spectrophotometer entire range of spectrum are recorded  Shows three-dimensional chromatogram  A plot of absorbance against elution time  The flow cell has a volume of 1–10 µL and a path length of 0.2–1 cm. Diode Array Detector PDA chromatogram  Refractive Index (RI) Detection Snell’s law of refraction – Sin i/Sin r It is a bulk property detector; Any change in its composition is reflected in the RI. The ability of a compound or solvent to deflect light provides a way to detect. The RI is a measure of molecule’s ability to deflect light in a flowing mobile phase in a flow cell relative to a static mobile phase contained in a reference flow cell. The amount of deflection is proportional to concentration. The RI detector is considered to be a universal detector but it is not very sensitive. Limitations: Commonly used water methanol solvent system RI changes considerably with temperature RI detection is generally incompatible with gradient elution. Not suitable for trace analysis  Fluorescence Detection The fluorescence detector is most sensitive detector than UV-Vis detectors It sense only detect those materials that will fluoresce or, by appropriate derivatization can be made to fluoresce Used in quantify and identify compounds and impurities in complex matrices at very low concentration levels Suitable for trace analysis/ genotoxic impurities Output :A plot of fluorescence intensity as a function of time. Less popular than the UV detectors since molecule should be fluoresce  Mass Spectroscopy (MS) An MS detector senses a compound eluting from the HPLC column first by ionizing it then by measuring it’s mass and/or fragmenting the molecule into smaller pieces that are unique to the compound. Mass spectrum is like a fingerprint and is quite unique to that compound. The MS detector used to identify the compound with library in application and also determine the quantity of molecule. MS detector is sensitive and used for trace analysis  Data Processer Data system/ processing : The computer controls all the modules of the HPLC instrument and converts the signal from the detector. Algorithms establish the time of elution (retention time) of the sample components (qualitative analysis) and the peak area ie amount of sample (quantitative analysis). Signals from the detector are processed based on the set of integration events and displays the chromatograms for easy to read and interpret. Availability of Function for process chromatographic data based on the application Computer with application available with many options like method development, determine system suitability, calibration, auto calculation etc. Also used to store, archive & back up of the data  Performance Qualification of HPLC Ensure the below parameters covered in the Performance Qualification as minimum, but not limited to; Component Parameter Acceptance Criteria Pump Pump Flow Accuracy 0.5ml (0.475 to 0.525) 5.0ml (4.75 to 5.25) Pump flow precision RT % RSD:NMT:0.50 Gradient composition in % 20, 40, 60, 80 + 2.0 Column oven Column Temperature Accuracy Column Oven: < 2.0 Column Temperature Stability Column Oven: < 1.0 Sample oven Sample temperature Accuracy Set temperature 4°C: > -2.00 and < 5.00 UV Detector Wavelength Accuracy (201nm to 209nm) 205 nm < 2 (241nm to 249nm) 245 nm < 2 (269nm to 277nm) 273 nm < 2 Noise and Drift Noise: < 0.040 mAU Drift: < 0.500 mAU/Hr Signal to Noise > 3000 Response Linearity (Resp.Factors) Correlation coefficient: > 0.99900 Lamp Intensity 1000 Sampler / Injector Precision % RSD for Area: < 1.0 % RSD for Height: < Injectors Volume Delivery - Linearity 2.00 Correlation coefficient: NLT:0.99 Injection Carryover Carryover for Area: < 0.20 Carryover for Area: < 0.40  Calibration of HPLC (UV) Component Calibration test Acceptance Criteria Pump Leak Test No leak Flow rate Accuracy 0.5ml (0.49 to 0.51) 1.0ml (0.98 to 1.02) 2.0ml (1.96 to 2.04) Flow stability RT % RSD:NMT:1.0 Gradient Delivery Accuracy in % 20, 40, 60, 80 ±2.0% Column oven Temperature Accuracy by Calibration of Column Oven: &Sampler Thermocouple and Air temperature 25°C/40°C/60°C + 2.0 UV Detector Wavelength Accuracy(266nm to 276nm) 271 to 273nm Dynamic Short-term Noise (Single to Noise:0.04 mAU or less Noise Ratio) Drift:5.0mAU/hr or less Response Linearity Correlation coefficient: NLT:0.99 Lamp energy Low intensity (> 200) Average intensity (> 5000) Highest intensity (> 10000) Sampler / Volume Precision % RSD :NMT 1.0 Injectors Volume Delivery – Linearity Correlation coefficient:NLT:0.99 Injector Carryover NMT 0.1  Technique & Method Selection Methods can be chosen based on solubility and molecular mass. Reversed-phase is appropriate for small molecules. Ion exchange is suitable for strong anion & cation Size Exclusion is appropriate for high molecular mass (>M 2000). Method Development RP- Basics Chemical nature and functional groups: Method Development RP- Basics  Column Chemistry Reverse phases: Phenyl : R = -C6H5, moderate C8 (octyl silane): R = -(CH2)7CH3, less hydrophobic C18/ODS: R = -(CH2)17CH3, hydrophobic Normal phases : Cyanopropyl [R = (CH2)3CN], less polar Diol: [R = -(CH2)2OCH2CH(OH)CH2OH], Amino : [R = -(CH2)3NH2], Dimethylamino, [R = -(CH2)3N(CH3)2] more polar Silica based columns have limited lifetime at pH levels below 2 or above 8.  Column Chemistry Silanol in the silica, is bonded with Octyl- or Octyldecyl group. The long-chain hydrocarbon groups are aligned parallel to one another and perpendicular to the surface of the particle, Present brush like, nonpolar hydrocarbon surface. Silica has limitation of stability over strong basic or alkaline pH, Silanol (Si-OH) encaped to increase the stability. Where required wider pH, Hybrid columns are used  Column and pH Organic–inorganic hybrid silica particle with ethylene bridge improves the stability to use wider pH range. Eg Propylene bridged bidentate C18 silane (Fig). Chromatographic columns with Graphitic carbon, alumina, titania, and zirconia are also available for wider pH tolerance. pH Cross-linked polymeric particles. for example, poly(methacrylate)s, and especially cross-linked poly(styrene), can withstand the full range of pH.  Chiral Columns Silicamatrix bonded to β- and γ - cyclodextrins via a small hydrocarbon chain /ether linkage. These cylindrically shaped ligands, which are oligosaccharides made of five to seven molecules of glucose, possess a hydrophobic internal cavity while the external part is hydrophilic. Central cavity is somewhat hydrophobic and the outer surface is hydrophilic. This gives them a selective permeability and it forms reversible diastereoisomer complexes at the surface and separates. – Eg.Cyclofructans (CFs) based stationary phases are commonly used in the normal separation mode of HPLC - to separate chiral primary amine enantiomers.  Selection of HPLC Columns Bonded Polarity Retention Comments Group mechanisms C18, C8, Nonpolar van der Waals does C8 does not retain hydrophobic C4 compounds as strongly as C18 Phenyl Nonpolar Hydrophobic and pi-pi Cyano Intermediate Hydrophobic, Resolves polar organic compounds dipole-dipole, and by reversed-phase or normal- pi-pi phase chromatography Amino Polar (NH2) Dipole-dipole and Normal-phase or ion-exchange Ionic ( NH3 H-bonding separations; separates ionic carbohydrates, polar organic compounds, and inorganic ions; reacts with aldehydes and ketones Bare silica Very polar H-bonding Normal-phase separations  Factors Affects the Performance pH of the mobile phase shuold be close to the pKa value of compound for better separation. Resolution is dependant on three variables, the column efficiency (N), capacity factor (k’) & selectivity (α). Selectivity is a measure of the relative retention of two adjacent peaks in a chromatogram Capacity factor is affected by changes in mobile phase, operating temperature, analyte retention characteristics and changes to the surface chemistry of the column. Increasing N increases resolution because peak width decreases. Decreasing k’ sharpens the peaks but decreases resolution. Increasing α increases resolution. capacity factor decreases with an increase in temperature according to the van’t Hoff equation Adjustment Allowed for HPLC Condition Property USP General Chapter 621 Ph.Eur. Gen. Chapter 2.2.46 Column length ±70% ±70% Particle size Reduction by 50% Reduction by 50% No increase No increase Internal diameter Can be adapted as long as the ±25% linear flow velocity remains the same Flow rate ±50% or more, provided the linear ±50% flow velocity remains the same Column temp. ±10 °C ±10 °C, maximum 60 °C pH -mobile phase ±0.2 units ±0.2 units (±1% for neutral subs)  Adjustment Allowed for HPLC Condition Property USP General Chapter 621 Ph.Eur. Gen. Chapter 2.2.46 Injection Reduction allowed as far as Reduction allowed as far as volume precision and detection limit precision and detection acceptable. No increase. limit acceptable. No increase. Salt conc. of the ±10%, as far as the allowed ±10% buffer change in pH value Composition of Minor components ±30%, if Minor components ±30%,if mobile phase not more than ±10% absolute not more than ±2% absolute (greater value accepted) * Wavelength Not permitted , can be Max ±3nm based on the validation *For gradient separation, a change of the mobile phase is not recommended  Analytical Method Validation Ensure that Analytical method used is validated to the above characteristics and suitable for its intended purpose (ICH Q2). Method should demonstrate Specificity, Precision (Repeatability Intermediate Precision, Reproducibility ), Linearity, Range, Accuracy, Robustness, Limit of Detection & Limit of Quantification as appropriate. If test method is as per monograph, ensure that Analytical method is verified for its suitability Eg USP General Chapter Any change in the test method /condition shall be within the allowable limit of Pharmacopeia methods (next page)  HPLC Application Qualitative Analysis – by comparing the retention time or volume of the sample to the standard against the sample. Retention time or relative retention time can be used for identification of eluted compound. Retention times are characteristic of the compounds they represent (but are not unique). Mass Detector gives the mass value to identify precisely. Quantitative Analysis- Pear area / Peak height of elution peak is proportional to the quantity / concentration. Peak response is based of the detector used. Method used for Quantitative Analysis shall be validated Type of estimation- Area normalization, Internal standard, Calibration and standard, Standard addition and etc  HPLC Application Pharmaceutical Applications Testing of Quality of raw material, in-process , intermediates, APIs and drug products Pharmaceutical quality control: Assay, related substances, content and, trace analysis like genotoxic, nitrosamine impurities at ppm level. To perform drug stability. Testing the content of pharmaceutical dosages form, content uniformity Phytochemical & nutraceuticals- plant extracts, Ginseng, herbal medicines Applications in Clinical Tests Bio-availability and bio-equivalency Toxicology, pharmacology, phatmacokinetics Urine analysis, analysis of blood, Bile acids, drug metabolites, urine extracts, estrogens etc Detection of endogenous Neuropeptides in extracellular fluid of brain etc.  HPLC Application Environmental Applications Detection of chemical compounds / contaminant in water and air quality Chemical Exposure in the workplace / environment Pesticides, herbicides, phenols, polychlorinated biphenyls (PCBs Applications in Forensics Identification & determination of abuse drugs in blood, urine etc. Eg. Cocaine, steroid, ketamine, amphetamine etc Quantification of Drugs, poisons, blood alcohol, narcotics. Forensic analysis like textile dyes, chemicals, etc Industrial Application: Identification & determination of cosmetics- Active ingredient content, purity, impurities and stability study. Analysis of Preservative, surfactants, propellants, dyes etc Organic chemicals like polymers (e.g. polystyrene, polyethylene) Artifcial sweeteners, antioxidants, aflatoxins, additives Thermally unstable compounds such as trinitrotoluene (TNT), enzymes  Tips for Good HPLC Practice  Always use only Ultra pure / Milli-Q water for HPLC analysis  Ultra pure HPLC water of 18MΩ resistivity  Do not use RO water/de-ionised water for HPLC analysis. It will have organic and in-organics impurities  If water contains impurities, it will have higher absorption and lead to poor baseline , drift , ghost peak and less accurate All reagents and solvents should be highest quality. HPLC grade reagents & solvents are high purity will have low UV absorbance Low grade solvent contain impurities to produce spurious peaks, poor peak , high baseline etc  Tips for Good HPLC Practice  Do not store HPLC columns in buffers. A buffer may precipitate inside the column, it will clog and affects the packing material.  Mobile phases with 100% or close to 100% buffer may lead to bacterial growth, which can block the column & frit and packing material.  Bacteria may also affect the analyte, and organic products from the dead bacteria may cause "ghost peaks" in chromatograms.  Do not store HPLC columns in solvents that degrade easily tetrahydrofuran (THF), triethylamine (TEA), trifluoroacetic acid (TFA).  Unstabilized THF can form peroxides which may degrade the column  All buffers should be washed out of the column before flushing with Acetonitrile.  Gradually start the column washing : Eg Starts the flow with 0.2 ml/min and increases gradually to 2.0 ml/min and continues for 20min to 30 min or as per procedure  Have Dedicated Columns for each Method / each product  Integration of Peaks Do not integrate any peak by manually. Integrate all the peaks or else as per procedure Always use same processing method for processing of blank, standard & sample chromatograms in case of Assay & related substances, etc. Verify the processing parameters like – Threshold, – Width, – System suitability, – Peak names etc. Save the processing method Re-integration: – Do not re-integrate the chromatograms without documenting. – Document the reason for reintegration.  Common Problems in HPLC Analysis System failures may occur during analysis due to – System over pressure – Leakage – Poor mobile phase dissolved gas, pH, solvent purity or composition, filtration, – Communication error – Failure of system suitability – Peak splitting/ negative peak – Spurious peak – Bracketing standard failure  Handling of Deviation/Failure  SOP shall be available and shall address the handling of Lab deviation/ incidents.  SOP shall define clearly the deviation/ incident , reporting investigation, CAPA and documentation  Record all the deviation/ incident happened in chromatographic analysis  Process all the injections including the invalid injection and report and store the data along with Raw data.  Do not omit any injection  Investigate the deviation/ incident and find the root cause for the failure.  Rectify the problem, take appropriate CAPA and document  Repeat complete sample set of injections in case of sample injection failure  References 1. Chemical Analysis: Modern Instrumentation Methods and Techniques- Francis and Annick Rouessac and Steve Brooks - John Wiley & Sons Ltd,. 2. Pharmaceutical Analysis David G. Watson 3. Analytical Chemistry for Technicians -John Kenkel 4. Analytical Chemistry - Gary D. Christian , Purnendu K. (Sandy) Dasgupta & Kevin A. Schug 5. Quantitative Chemical Analysis- Daniel C. Harris 6. Vogel’s – Quantitative Chemical Analysis- 6th edition 7. Principles of Instrumental Analysis- 6th Edition- D.A. Skoog et al 8. United States Pharmacopeia 9. European Pharmacopeia About Author: Dr. A. Amsavel, born at Begarahalli, Dharmapuri-Dist, Tamil Nadu, India. Completed his M.Sc. in Dept of Analytical Chemistry, University of Madras. B.Ed. in Annamalai University and Ph.D in Anna University, Chennai. Worked as Lecturer and also worked in various Chemical & Pharmaceutical Industries for the past 34 years. Presently working as Assistant Vice President- Quality at Malladi Drugs & Pharmaceuticals Ltd. 59